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Journal ofFood Protection Vol. 42. No.3. Pages 218-221 (i\1arch.1979) Copyright 1979, International Association of , , and Environmental Sanitarians

Types of and Shelf- of Evacuated Dioxide­ Injected and Ice-Packed Broilers

J. S. BAILEY1,2•, J. 0. REAGANl, J. A. CARPENTERl, G. A. SCHULERl andJ. E. THOMSON2

Food Science Department, University ofGeorgia, Athens, Georgia 30602, and Richard B. Russell Agricultural Research Center, United States Department ofAgriculture, Science and Education Administration, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/3/218/1649927/0362-028x-42_3_218.pdf by guest on 27 September 2021 P.O. Box 5677, Athens, Georgia 30604

(Received for publication June 29, 1978)

ABSTRACT microflora on spoiled stored pork were or Broiler carcasses were packed in low-permeability film bags that . Clark and Lentz (8) found that in

were evacuated, injected with C02 , then held at 2 C. Broilers were -packaged held at 5 C, the Pseudomonas examined microbiologically and for off-odor to determine the shelf-life and the - group were inhibited and types of bacteria. Vacuum level did not significantly affect by 15% C02 • Shank and Lundquist (29) showed that the bacterial counts. Broilers stored in 65"7o C0 had a 1-day longer 2 spoilage of vacuum-packaged cured involved lactic shelf-life than those held in 20o/o C02 and about a 5-day longer shelf-life than ice-packed broilers. Spoiled carcasses from either 20 or acid bacteria almost exclusively. Patterson and Gibbs

65% C02 packages had an acid-sour off-odor, and more than 90% of (23) found that lactobacilli were the predominant the bacteria present were . Ice-packed broilers had the spoilage bacteria on beefthat was vacuum packaged and typical putrid off-odor at spoilage, and more than 95% of the bacteria held at 0-2 C. Pierson et al. (24) found that 95% of the were non-pigmented Pseudomonas. bacteria on anaerobically packaged beef were lacto­ . Lactobacillus and other bacteria whose

growth is not significantly inhibited by C02 have a direct Kraft (19) listed 24 genera of bacteria as isolates from bacteriostatic effect on many gram-negative bacteria (14) poultry and Cox (9) found one additional . Only a and on three strains of Pseudomonas and one of few genera, however, affect the acceptability and typhimurium (25). shelf-life of poultry . Most observers found many In this study we aimed to determine the effect of C02 different types of bacteria at the of slaughter, but on and vacuum on types of bacteria on broilers in carcasses that were held aerobically at 0-5 C the commercially feasible, bulk shipping packs of low Pseudomonas or Achromobacter groups predominated permeability film, and to determine the effect of this at the time of spoilage (1,3,5,15,22.23). microf1ora on shelf-life and olfactory quality of broilers. Coyne (12), Haines (16), Ogilvy and Ayres (22) and Scott (28) found that the two principal spoilage Pseudomonas and Achromobacter were MATERIALS AND METHODS inhibited by C02 • Average generation time for A "Conofresh 4000" packaging system (Continentia! Forest Pseudomonas apparently was reduced as a logarithmic Industries - Corrugated, Greenwich, CT)4 was used to package 560 function of C02 level (22). Haines (16) and King and broiler carcasses in a simulated commercial evacuated, C02 -flushed bulk pack. Chilled broilers were taken from the drip line at a Nagel (17) found that C02 levels of 20 to 70% doubled the generation time for Pseudomonas if other environ­ commercial processing and placed four to a plastic film bag (7 cc/100 in2 , permeability); then bags were placed into a wax coated mental factors were constant. Thomson (32) found that corrugated box for insertion into the packaging chamber. The chamber psychrotrophs were inhibited by 10 and 20o/oC02 • was evacuated to 10, 17 or 24 inches of Hg. was The predominant spoilage on beef injected into a film bag to either 20 or 65%, and the bag was sealed. stored in air were Pseudomonas and Achromobacter Percentage of C02 in each bag was not measured directly, but was (2,18,26). Gardner et al. (13) found that 96% of the based on extrapolation from the known pressure applied by the C02 injection mechanism; the pressure required to produce each C02 content with this packaging equipment had been determined 1 University ofGeorgia. previously. The packs were held at 2 C and sampled for 2 Russell Agricultural Research Center. microorganisms and for off-odor after S, 9, 12, 15 and 18 days of 3 The eighth edition of Bergey's Manual of Determinative now classifies Achromobacter as Acinetobacter. storage. Controls consisted of broilers that were packed in ice and held 4 Mention of specific brand !Ulmes does not imply endorsement by the in wax-coated corrugated boxes and stored at 2 C. Controls were also authors or the U.S. Department of to the exclusion of sampled microbiologically and for off-odor after 5, 9, 12, 15 and 18 others not mentioned. days of storage. All treatments were duplicated. BACTERIA AND SHELF-LIFE OF C02-PACKED BROILERS 219

For total plate counts a 12.3-cm2 area of breast was swabbed 13----El CONTROLS 8 with calcium alginate swab which then was dissolved in a sterile 9.9-ml • 20% C02 1 o/o sodium citrate blank. Appropriate serial dilutions were made, • ~ 65% C02 plated in duplicate on Plate Count (Difco) and plates were 7 / incubated for 48 h at 30 C. Counts were expressed as log1 0 /em'. After sampling, carcasses were discarded and a new bulk pack was opened on each sampling day. Carcasses were evaluated for off-odor when packs were opened for "':E 6 ~~ microbiological sampling. Sampling was terminated when the carcasses u became spoiled, as determined by strong off-odor. Strong off-odor had Q: IJJ become apparent on the carcass at a count of log 6.5 which, therefore, a.. 5 was defined as spoilage. r I- _;;r-~ All data were analyzed by the Statistical Analysis System of Barr and z :::> Goodnight (6). Significance was determined by the F-test and a 0 4 G-d difference was considered significant at the 5o/o level of probability. u ~~ A total of 375 isolated colonies were picked from countable plates <{ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/3/218/1649927/0362-028x-42_3_218.pdf by guest on 27 September 2021 and generically classified with the identification schemes of Q: Biochemical Tests for Identification of Medical Bacteria 1)0), and IJJ 3 Bergey's Manual of Determinative Bacteriology (7). Thirty colonies I-u were picked from two plates each day that packs were opened for <{ m evaluation. For control samples, 15 colonies were picked from duplicate 2 plates on sampling days. A statistical randomizing procedure 0 (!) employing a numerical grid (10) and a random numbers table (30) was 0 used to obtain a representative sample ofthe colonies from each plate. _J Colonies were transferred to brain heart infusion (BHI) broth (Difco) and incubated at 25 C for 48 h. BHI plates were streaked for and then incubated at 25 C for 18 to 24 h. Isolated colonies were 0 picked. checked for purity and then tested for: gram reaction and 3 6 9 12 15 18 morphology, , and and reaction. We then did the additional tests which were necessary for generic identification. The biochemical characteristics that identified each genus were as follows: DAYS OF STORAGE IN BULK PACK (a) - gram-positive, catalase-positive cocci, no growth in 15% NaCI, no growth in 40o/o bile and Voges Proskauer-negative; (b) Figure 1. Total aerobic bacterial counts of broilers packaged in - gram-positive, catalase-positive cocci, growth in 15o/o vacuum in bulk packs containing 20 and 65o/o CO, and of ice-packed NaCI. growth in 40o/o bile and Voges·Proskauer-positive; (c) controls. All were stored at 2 C. Lactobacillus- gram-positive, catalase-negative non-sporeformingrods, no reduction of , no true branching (a representative sample of before birds stored in 20% C02 and about 5 days before Lactobacillus isolates was confirmed by use of Lactobacillus- 50 strips - birds stored in 65%C02 • At the time of spoilage, control API); (d) - gram-positive, catalase-positive, sporeforming rods; carcasses had the typical putrid off-odor that was {e) thermosphactum -gram-positive, catalase-positive, characterized by Ayres et al. (3). nonmotile rods, -positive and growth on STAA agar (14); If! - gram-positive, catalase-positive, non-sporeforming The bacterial population of evacuated packaged rods, H 2S-negative, gelatin liquefaction-positive; (g) Pseudomonas broilers with C02 injected into packages was pre­ jluorescens - gram-negative, catalase-positive rods, oxidase-positive, dominantly Lactobacillus (Tables 1, 2). Packs containing motile, slow growth at 37 C, no growth at 41 C, no reduction of nitrate, 20o/o C02 showed a mixed microflora and Lactobacillus no of dextrose, fluorescent pigment produced; (h) constituted 53 o/o of the bacteria on day 5 of storage; non-pigmented Pseudomonas - gram-negative, catalase-positive rods, motile, oxidase-positive, aerobic acid production from dextrose, no Lactobacillus constituted not less than 97 o/o of the anaerobic fermentation of dextrose, no pigment produced; {z) microflora thereafter (Table 1). After 18 days of Acinetobacter (formerly Achromobacter) gram-negative, catalase· storage, broilers were spoiled and 100o/o of the bacterial positive rods, oxidase-negative, -negative, H2 S-negative.

RESULTS AND DISCUSSION TABLE 1, Percent of isolates recovered from broilers held in vacuumized shipping packs containing 20o/o C0 and stored at 2 C. Bacterial counts on carcasses were significantly lower 2

in vacuum-treated packages flushed with C02 than on controls at 15 and 18 days (Fig. 1). Counts on carcasses in Micrococcus sp. 65%C02 were significantly lower after 15 days. Vacuum Staphylococcus sp. 3 levels did not significantly affect bacterial counts Lactobacillus sp. 97 97 100 100 throughout these experiments. At the time of spoilage, Bacillus sp. 3 off-odor of carcasses in C0 was acid-sour. Ogilvy and Microbacterium 2 thermosphactum Ayres (22), Shank and Lundquist (29) and Sutherland et Coryneform sp. 17 al. (31) also found a characteristic acid-sour off-odor Pseudomonas.fluorescens 13 when were the predominant spoilage Non-pigmented Pseudomonas bacteria on meats. Acinetobacter sp. 3 Control birds had the highest counts on all sampling Gram-negative days and spoiled in 14 days, which was about 4 days 220 BAILEY ET AL.

TABLE 2. Percent of isolates recovered from broilers held in grew on our stored broiler carcasses. The predominant vacuumized shipping packs containing 65% C0 and stored az2 C. 2 spoilage bacteria on aerobically stored ice-packed Days of storage broilers were non-pigmented Pseudomonas species and Isolates on vacuum-packaged, C02 -injected packs of broilers Micrococcus sp. were Lactobacillus species. This difference resulted in Staphylococcus sp. 13 Lactobadllus ;,p. 77 97 83 an extended shelf-life and an acid-sour type of spoilage, Bact1lus sp. rather than the putrid type of spoilage in broilers that Microbacterium were ice-packed. As an alternative to the commonly thermosphactum 3 10 Coryneform sp. 10 3 used ice-pack system of packing broilers for transport, Pseudomonas.fluorescens 13 7 10 the bulk vacuum-C02 packaging system offers ad­ Non-pigmented vantages that include longer product shelf-life and Pseudomonas Acinetobacter sp. greater economy, the ability to ship mixed meat Gram-negative species 3 loads, reduction of the short weight problem and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/3/218/1649927/0362-028x-42_3_218.pdf by guest on 27 September 2021 Moldsp. 3 elimination of the use of ice in shipping.

population sampled were Lactobacillus. Packs with 65 o/o ACKNOWLEDGMENT

C02 also showed a mixed microflora on day 5 of storage, We thank Gary Hamed of the Continental Can Company for making and 47o/oofthe bacteria were Lactobacillus (fable 2). On possible a grant to support this project. We thank Janice Carpenter and day 9 of storage, 77% were Lactobacillus, and this Elizabeth Bower for their technical assistance and Con Agra poultry, Dalton, Georgia for their assistance and use oftheir plant and personnel proportion remained high until18 days of storage, when in preparation of the samples. birds were spoiled and 90 o/o of the bacteria were Lactobacillus. These findings confirmed other studies REF'ERENCES (14,22,21) in which lactobacilli predominated on poultry 1. Arafa, A. S., and T. C. Chen. 1975. Effect of vacuum packaging on or meat stored in C02 • microorganisms on cut-up chickens and in chicken products. J. The microflora was mixed on control carcasses Food Sci. 40:50-52. through day 12 of storage, when non-pigmented 2. Ayres, J. C. 1960. Temperature relationships and some other characteristics of the microbial developing on refrigerated Pseudomonas accounted for 40% of the bacteria present beef. Food Res. 25:1-18. (fable 3). The non-pigmented Pseudomonas made up 3.Ayres, J. C., W. S. Ogilvy. and G. F. Stewart. 1950. Post-mortem 93 o/o of the bacterial population by day 15 of storage when changes in stored meats. 1. Microorganisms associated with develop­ the carcasses were spoiled and 1000'/o by day 18 when a ment of slime on eviscerated cut-up poultry. Food Technol. had formed on the surface of the carcasses. 4:199-205. 4. Barnes, E. M., and C. S. Impey. 1968. Psychrophilic spoilage McMeekin (2]) also showed that group II non-pigmented bacteria of poultry. J. Appl. Bacteriol. 31:97-107. Pseudomonas (odor producers) outgrew group I Pseudo· 5. Barnes. E. M., and M. J. Thornley. 1966. The spoilage flora of monas (fluorescent) when chicken meat was held at 2 C, eviscerated chickens stored at different temperatures. J. Food and Barnes and Impey (4) and Cox et al. (11) found that Techno!. I: 113-119. non-pigmented Pseudomonas produced a more intensive 6. Barr, A. I., and J. H. Goodnight. 1972. A users guide to the statistical analyses system. Student Supply Store, North Carolina off-odor than other bacteria present and were the State University, Raleigh, NC. predominant present when aerobically­ 7. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual stored poultry reached spoilage. of determinative bacteriology, 8th ed. The Williams and Wilkins Packaging environments, whether ice-packed or Co .. Baltimore, MD. vacuum-packed. determined the types of bacteria that 8. Clark, D. S., and C. P. Lentz. 1973. Use of mixtures of carbon dioxide and for extending shelf-life of prepackaged fresh beef. Can. Institute Food Sci. Technol. J. 6:194-196. 9. Cox, 1'i. A. 1975. Isolation and identification of a genus, TABLE 3. Percent o.fisolates recovered from ice· packed broilers held Chrumobacterium, not previously found on processed poultry. at 2 C. Appl. :vticrobiol. 29:864. 10. Cox, N. A., A. 1. Mercuri. B. J, Juven, J. E. Thomson, and V. Days of storage Chew. 1974. Evaluation of succinic acid and heat to improve the Isolates 5 9 12 15 lS microbiological quality of poultry meat. J. Food Sci. 39:958-987. l\Jicrococcus sp. 20 13 27 11. Cox, N, A .. B. J.Juven, J. E. Thomson, A. J. Mercuri, and V. Chew. Staphylococcus sp. 13 27 20 1975. Spoilage odors in poultry meat produced by pigmented and Lactobacillus sp. 33 20 7 non-pigmented Pseudomonas. Poultry Sci. 54:2001-2006. Bacillus sp. 12. Coyne, F. P. 1933. The effect of carbon dioxide on . Microbacterium thermosphactum Royal Society of London Proceedings. Series B. Bioi. Sci. Coryn~(orm sp. 13 33 7 7 113:196-216. 13. Gardner. G. A., A. W. Carson, and J. Patten. 1967. Bacteriology of Non-pigmented prepacked pork with reference to the gas composition within the Pseudomonas 20 7 40 93 100 pack. J. Appl. Bacteriol. 30:321-333, Acinetobacter sp. 14. Gilliland, S. C., and M. L. Speck. 1977. Antagonistic action of Gram-negative species Lactobacillus acidophilus toward intestinal and foodborne patho­ Mold sp. gens in associative cultures . .T. Food Prot. 40:820-823. BACTERIA AND SHELF-LIFE OF C02-PACKED BROILERS 221

15. Gunderson, M. R., H. W. McFadden, and T. S. Kyle. 1954. The Microbiological, sensory and pigment changes of aerobically and bacteriology of commercial poultry processing. Burgess Publishing anaerobically packaged beef. Food Techno!. 24:1171-1175. Co., Minneapolis, MN. 25. Raccach. M., and R. C. Baker. 1978. The role of lactic acid 16. Haines, R. B. 1933. The influence of carbon dioxide on the rate of bacteria as an antispoilage and safety factor in cooked, multiplication of certain bacteria, as judged by viable counts. J. mechanically de boned poultry meat. J. Food Prot. 41:703-705. Soc. Chern. Ind. 52:13-17. 26. Rodger, R. E., and C. S. McCleskey. 1957. Bacteriological quality 17. King, D. A .. and C. W. NageL 1967. Growth inhibition of a of ground beefin retail markets. Food Techno!. 11:318-323. Pseudomonas by carbon dioxide. J. Food Sci. 32:575·579. 27. Roth, L. A., and D. S. Clark. 1975. Effect of lactobacilli and 18. Kirsch, R. H., F. E. Berry, C. L. Baldwin, and E. M. Foster. 1952. carbon dioxide on the growth of Microbacterium thermosphactum The bacteriology of refrigerated ground beef. Food Res. 17:495-503. on fresh beef. Can. J. Microbiol. 21:629-632. 19. Kraft, A. A. 1971. of poultry products. J. Milk Food 28. Scott, W. J. 1938. The growth of microorganisms on ox muscle. III. Techno!. 34:23-29. The influence of 10o/o carbon dioxide on rate of growth at - 1"C. 20. MacFaddin, J. F. 1976. Biochemical tests for identification of Australia, Commonwealth Council Sci. Ind. Research, 11:266-267. medical bacteria. The Williams and Wilkins Co., Baltimore. MD. 29. Shank, J. L., and B. R. Lundquist. 1%3. The effect of packaging 21. McMeekin, T. A. 1975. Spoilage association of chicken breast conditions on the ba..ieriology, color, and flavor of table-ready muscie. Appl. Microbiol. 29:44-47. meats. Food Technol.l7:1163-1166. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/42/3/218/1649927/0362-028x-42_3_218.pdf by guest on 27 September 2021 22.0gilvy, W. S .. and J. C. Ayres. 1951. Post-mortem changes in 30. Snedecor, G. W. (ed). 1956. Statistical methods. Sth ed, Iowa State stored meats. II. The effect of atmospheres containing carbon College Press, Ames, Iowa. dioxide in prolonging the storage life of cut-up chickens. Food 31. Sutherland, J.P., J. T. Patterson. and J. G. Murray. 1975. Changes Techno!. 5:97·103. in the microbiology of vacuum-packaged beef. J. Appl. Bacteriol. 23. Patterson, J. T., and P. A. Gibbs. 1977. Incidence and spoilage 39:227-237. potential of isolates from vacuum-packaged meat of high pH value. 32. Thomson, J. E. 1970. Microbial counts and rancidity of fresh fryer J. Appl. Bacteriol. 43:25-38. chickens as affected by packaging materials, storage atmosphere 24. Pierson, M.D., D. L. Collins -Thompson, and Z. J. Ordal. 1970. and temperature. Poultry Sci. 49:1104-1109.